Crystalline forms of 3-substituted 1,2,4-oxadiazole

ABSTRACT

The invention relates to crystalline forms of a 3-substituted 1,2,4-oxadiazole compound, methods of their preparation, and related pharmaceutical preparations thereof. The invention also relates to preparations suitable for pharmaceutical, veterinary, and agriculturally-relevant uses.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the § 371 National Stage of PCT/IB2018/057840, filedOct. 10, 2018, which claims the benefit of priority to IndianProvisional Patent Application serial number 201741036169, filed Oct.11, 2017, the contents of each of which are hereby incorporated byreference herein in their entirety.

STATEMENT OF JOINT RESEARCH AGREEMENT

The inventions disclosed herein were made as a result of activitiesundertaken within the scope of a joint research agreement betweenAurigene Discovery Technologies Ltd., and Curis, Inc., which agreementwas in effect on or before the effective filing date of the claimedinvention.

BACKGROUND

The immune system in mammals regulates the activation and inactivationof lymphocytes through various mechanisms during and after an immuneresponse. Among these mechanisms, there are mechanisms that specificallymodulate the immune response as and when required.

3-substituted 1,2,4-oxadiazole compounds act as immunomodulators. Thus,3-substituted 1,2,4-oxadiazole compounds can be used in the treatment ofcancer, immune disorders, immunodeficiency disorders, inflammatorydisorders, infectious diseases, and transplant rejection.

Given the therapeutic benefits associated with 3-substituted1,2,4-oxadiazole compounds, there is a need for improved compositions ofthese compounds. Further, there is a need for improved methods forpreparing and formulating 3-substituted 1,2,4-oxadiazole compounds.

SUMMARY

One aspect of the invention relates to a crystalline compound having thestructure of formula (I),

Another aspect of the invention relates to methods for preparing thecrystalline compounds of formula (I).

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising acrystalline compound of formula (I), and one or more pharmaceuticallyacceptable excipients. In certain embodiments, the pharmaceuticalpreparations may be for use in treating or preventing a condition ordisease as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the XRPD patterns of formula (I) Form 1.

FIG. 2 shows overlay XRPD patterns of formula (I) Form 2 from example 2Dand Example 2E

FIG. 3 shows the thermogravimetric analysis and differential scanningcalorimetry thermogram of Form 1.

FIG. 4 shows the thermogravimetric analysis and differential scanningcalorimetry thermogram of Form 2.

FIG. 5 shows a comparison of XRPD patterns of formula (I) Form 2 at 25°C., 60% relative humidity (RH); 40° C., 75% RH; and 30° C., 56% RH after7-8 days compared to a Form 2 reference sample in solid form.

FIG. 6 shows a comparison of XRPD patterns of formula (I) Form 2 inwater at 20° C.; in water at 30° C.; and Form 2 after solubilitytesting.

FIG. 7A shows the asymmetric unit of the formula (I) Form 2 singlecrystal.

FIG. 7B shows a proposed proton transfer to form a zwitterion in theformula (I) Form 2 crystal.

FIG. 8 shows the XRPD patterns of amorphous formula (I).

DETAILED DESCRIPTION

In certain embodiments, the invention provides a crystalline compoundhaving the structure of formula (I),

In some embodiments, the invention provides a crystalline compoundhaving the structure of formula (I) could also be written by showing allof the atoms,

In certain embodiments, a crystalline compound of formula (I) issolvated. In certain such embodiments, the crystalline compound offormula (I) is a hydrate (e.g., a monohydrate or a dihydrate). Incertain particular embodiments, the crystalline compound of formula (I)is a monohydrate. In certain particular embodiments, the crystallinecompound of formula (I) is a dihydrate.

Any crystalline compound described herein may be used in the manufactureof a medicament for the treatment of any diseases or conditionsdisclosed herein.

In certain embodiments, the compounds of the present invention canassemble into more than one crystal formation. In an exemplaryembodiment, the crystalline compound having the structure of formula (I)exists as “Form 1”, “Form 2”, or a mixture thereof, as described indetail below. These different forms are understood as “polymorphs”herein.

In certain embodiments, the polymorph of the crystalline compound ischaracterized by powder X-ray diffraction (XRD). θ represents thediffraction angle, measured in degrees. In certain embodiments, thediffractometer used in XRD measures the diffraction angle as two timesthe diffraction angle θ. Thus, in certain embodiments, the diffractionpatterns described herein refer to X-ray intensity measured againstangle 2θ.

In certain embodiments, a crystalline compound of formula (I) issolvated.

In some embodiments, a crystalline compound of formula (I) is solvatedwith water and is a hydrate. In certain embodiments, the crystallinehydrate of the compound of formula (I) is a monohydrate or a dihydrate.In other embodiments, the crystalline hydrate of the compound of formula(I) is a monohydrate. In other embodiments, the crystalline hydrate ofthe compound of formula (I) is a dihydrate.

In certain embodiments, a crystalline hydrate of the compound of formula(I) has 2θ values 8.4±0.2, 13.6±0.2, 16.5±0.2, 16.8±0.2, 21.4±0.2, and28.4±0.2. In further embodiments, the crystalline hydrate has 2θ values8.4±0.2, 13.6±0.2, 16.5±0.2, 16.8±0.2, 19.3±0.2, 20.4±0.2, 21.4±0.2, and28.4±0.2. In yet further embodiments, the crystalline hydrate has 2θvalues 8.4±0.2, 13.6±0.2, 16.5±0.2, 16.8±0.2, 19.3±0.2, 19.9±0.2,20.4±0.2, 21.4±0.2, 24.5±0.2, 26.5±0.2, and 28.4±0.2. In furtherembodiments, the crystalline hydrate has 2θ values 8.4±0.2, 11.5±0.2,13.6±0.2, 16.5±0.2, 16.8±0.2, 19.3±0.2, 19.9±0.2, 20.4±0.2, 21.4±0.2,21.8±0.2, 24.5±0.2, 26.5±0.2, 27.5±0.2, 28.0±0.2, 28.4±0.2, 30.0±0.2,and 32.4±0.2. In some embodiments, the crystalline hydrate has 2θ valuesselected from the following peaks listed Table 1±0.2.

TABLE 1 Exemplary peaks of Form 1 Pos. Height FWHM Left d-spacing Rel.Int. [°2Th.] [cts] [°2Th.] [{acute over (Å)}] [%] 6.818730 477.0624000.153504 12.96356 9.84 8.429180 1106.154000 0.230256 10.49006 22.839.996699 162.963200 0.179088 8.84842 3.36 10.641450 181.680600 0.2046728.31371 3.75 11.473900 856.673700 0.179088 7.71235 17.68 13.5866204846.171000 0.179088 6.51746 100.00 16.523190 2235.245000 0.1790885.36517 46.12 16.841620 1711.580000 0.153504 5.26444 35.32 17.64259099.168370 0.153504 5.02720 2.05 18.833270 580.986700 0.127920 4.7119711.99 19.308990 1653.407000 0.179088 4.59694 34.12 19.940700 1511.7410000.153504 4.45272 31.19 20.379910 1426.443000 0.179088 4.35774 29.4320.640480 1063.284000 0.127920 4.30331 21.94 20.981490 582.9376000.153504 4.23413 12.03 21.395090 1507.023000 0.153504 4.15321 31.1021.751640 776.502400 0.153504 4.08593 16.02 22.616420 99.647250 0.2046723.93161 2.06 23.625170 188.599900 0.153504 3.76598 3.89 24.5235001372.566000 0.179088 3.63002 28.32 24.824960 695.664600 0.102336 3.5866214.35 25.319510 269.242600 0.179088 3.51768 5.56 26.486100 1097.9680000.204672 3.36533 22.66 27.046260 201.561100 0.179088 3.29689 4.1627.509350 900.113200 0.204672 3.24243 18.57 28.032180 818.2289000.153504 3.18313 16.88 28.357680 1216.173000 0.179088 3.14733 25.1029.284110 65.256650 0.153504 3.04984 1.35 30.078100 824.492300 0.2302562.97112 17.01 30.808680 118.232400 0.204672 2.90231 2.44 31.530650388.319600 0.153504 2.83748 8.01 32.440480 869.745500 0.204672 2.7599517.95 33.044710 264.978200 0.127920 2.71085 5.47 33.486020 328.8151000.179088 2.67613 6.79 34.329380 233.555300 0.102336 2.61229 4.8235.275910 345.531500 0.179088 2.54434 7.13 36.461080 277.000200 0.1023362.46431 5.72 37.629320 368.294900 0.179088 2.39044 7.60 38.089640238.710400 0.204672 2.36261 4.93 38.729290 317.873000 0.153504 2.325056.56

In certain embodiments, a crystalline hydrate of the compound of formula(I) has an XRD pattern substantially as shown in FIG. 1, labeled Form 1.

In some embodiments, the crystalline hydrate has 2θ values 12.9±0.2,13.5±0.2, 15.7±0.2, 17.0±0.2, 29.7±0.2, and 33.7±0.2. In furtherembodiments, the crystalline hydrate has 2θ values 12.9±0.2, 13.5±0.2,15.7±0.2, 17.0±0.2, 20.3±0.2, 28.9±0.2, 29.7±0.2, and 33.7±0.2. In yetfurther embodiments, the crystalline hydrate has 2θ values 12.9±0.2,13.5±0.2, 15.7±0.2, 17.0±0.2, 19.6±0.2, 20.3±0.2, 26.2±0.2, 28.9±0.2,29.7±0.2, and 33.7±0.2. In still further embodiments, the crystallinehydrate has 2θ values 12.9±0.2, 13.5±0.2, 15.7±0.2, 17.0±0.2, 19.1±0.2,19.6±0.2, 20.3±0.2, 21.1±0.2, 21.4±0.2, 26.2±0.2, 27.2±0.2, 28.9±0.2,29.7±0.2, 32.2±0.2, and 33.7±0.2. In some embodiments, the crystallinehydrate has 2θ values selected from the following peaks listed Table2±0.2.

TABLE 2 Exemplary peaks of Form 2 Pos. Height FWHM Left d-spacing Rel.Int. [°2Th.] [cts] [°2Th.] [{acute over (Å)}] [%] 10.926180 397.1336000.102336 8.09770 19.80 11.592880 174.246700 0.127920 7.63345 8.6912.933320 570.548600 0.102336 6.84516 28.45 13.459840 2005.2570000.102336 6.57856 100.00 15.662360 1080.314000 0.102336 5.65806 53.8716.071480 201.940400 0.102336 5.51494 10.07 16.951470 1230.6260000.127920 5.23057 61.37 19.134270 1076.042000 0.127920 4.63852 53.6619.553070 1440.614000 0.127920 4.54011 71.84 20.306040 1175.6040000.204672 4.37342 58.63 21.053740 714.116700 0.102336 4.21977 35.6121.422720 517.599900 0.102336 4.14791 25.81 22.172010 165.2196000.102336 4.00940 8.24 22.869490 92.001400 0.153504 3.88868 4.5924.193950 163.135600 0.127920 3.67871 8.14 24.523350 238.576700 0.1023363.63004 11.90 24.789210 371.732200 0.102336 3.59171 18.54 25.095920276.245600 0.102336 3.54851 13.78 25.791380 281.323200 0.102336 3.4543814.03 26.153440 745.307100 0.102336 3.40737 37.17 26.402310 226.2782000.076752 3.37582 11.28 27.224340 576.143400 0.153504 3.27572 28.7328.230660 218.307800 0.102336 3.16120 10.89 28.862240 803.4570000.127920 3.09345 40.07 29.715230 554.454300 0.179088 3.00657 27.6530.204860 251.635200 0.076752 2.95894 12.55 31.028680 198.2701000.102336 2.88223 9.89 32.232440 414.918300 0.127920 2.77728 20.6933.668980 788.804300 0.153504 2.66200 39.34 34.180000 83.372570 0.3070082.62336 4.16 36.002300 76.087860 0.153504 2.49465 3.79 37.016770103.663100 0.204672 2.42858 5.17 38.371850 39.207050 0.307008 2.345881.96 38.980620 67.341840 0.204672 2.31063 3.36

In certain embodiments, a crystalline hydrate of the compound of formula(I) has an XRD pattern substantially as shown in FIG. 2, labeled Form 2.

In certain embodiments, the crystalline hydrate of the compound offormula (I) is a monohydrate. In certain embodiments, the crystallinehydrate of the compound of formula (I) contains about 0.9, about 1.0,about 1.1, or about 1.2 molecules of water to one molecule of thecompound of formula (I). In certain embodiments, the crystalline hydrateof the compound of formula (I) by differential scanning calorimetry(DSC) shows one or two overlapping endotherms with a peak temperaturefrom about 115° C. to about 145° C. before melting/decomposition. Insome embodiments, the crystalline hydrate of the compound of formula (I)shows one or two overlapping endotherms with a peak temperature in therange selected from about 116° C. to about 140° C. beforemelting/decomposition. In some embodiments, the crystalline hydrate ofthe compound of formula (I) shows one or two overlapping endotherms witha peak temperature in the range selected from about 120° C. to about140° C., about 125° C. to about 135° C., and about 126° C. to about 133°C. by DSC before melting/decomposition. See FIG. 3, labeled Form 1.

In certain embodiments, the crystalline hydrate of the compound offormula (I) is a dihydrate. In certain embodiments, the crystallinehydrate of the compound of formula (I) contains about 1.8, about 1.9,about 2.0, about 2.1, or about 2.2 molecules of water to one molecule ofthe compound of formula (I). In certain embodiments, the crystallinehydrate of the compound of formula (I) by differential scanningcalorimetry (DSC) shows two endotherms with a peak temperature of thefirst endotherm in the range selected from about 25° C. to about 65° C.,30° C. to about 60° C., about 40° C. to about 55° C., and about 45° C.to about 50° C.; and a peak temperature of the second endotherm in therange selected from about 60° C. to about 85° C., about 65° C. to about80° C., and about 70° C. to about 75° C. before melting/decomposition.See FIG. 4, labeled Form 2.

In some embodiments, the crystalline hydrate of the compound of formula(I) shows melting/decomposition with an onset temperature in the rangeselected from about 178° C. to about 190° C., about 182° C. to about186° C., and about 183° C. to about 185° C. by DSC.

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a crystalline compound of formula (I) and one ormore pharmaceutically acceptable excipients. In certain embodiments, thepharmaceutical composition is selected from tablets, capsules, andsuspensions.

In certain embodiments, the pharmaceutical composition comprises thesalt of the compound of formula (I) and one or more pharmaceuticallyacceptable excipients.

Defintions:

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

As used herein, the term “hydrate” refers to a complex formed by thecombining of Compound of formula (I) and water. The term includesstoichiometric as well as non-stoichiometric hydrates.

As used herein, the term “solvate” refers to a complex formed by thecombining of Compound of formula (I) and a solvent.

As used herein, “therapeutically effective amount” refers to an amountthat is sufficient to effect treatment, when administered to a mammal inneed of such treatment. The therapeutically effective amount will varydepending upon the subject being treated, the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

The term “substantially pure”, as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that it contains lessthan 10% of any other compound, or an alternative polymorph of thecrystalline form. Preferably, the crystalline polymorph is greater than95% pure, or even greater than 98% pure.

The phrase “substantially as shown in FIG.” refers to an X-ray powderdiffraction pattern with at least 50%, or at least 60%, or at least 70%,or at least 80%, or at least 90%, or at least 95% or at least 99% of itspeak appears in FIG.

As used herein, the term “about” when referring to a number or anumerical range means that the number or numerical range referred to, isan approximation within experimental variability (or within statisticalexperimental error), and thus the number or numerical range may varyfrom, for example, between 1% and 15% of the stated number or numericalrange.

Methods of Making the Crystalline Forms of the Compound of Formula (I)

In certain embodiments, the invention relates to a method for preparinga crystalline compound having the structure of formula (I), comprising:

-   a) providing a mixture comprising a compound of formula (I) and a    solvent; and-   b) crystallizing the compound of formula (I) from the mixture    comprising the compound of formula (I).

In certain embodiments, the mixture comprising the compound of formula(I) and the solvent is a reaction mixture.

In certain embodiments, the mixture comprising the compound of formula(I) is a solution. In certain embodiments, the solution comprises acompound of formula (I) dissolved in a solvent. In some embodiments, thesolution comprises a crude solid material comprising the compound offormula (I) dissolved in a solvent. In some embodiments, the solutioncomprises a reaction mixture.

In certain embodiments, the mixture is a slurry or a suspension. Incertain embodiments, the slurry or the suspension comprises crude solidmaterial comprising the compound of formula (I).

In certain embodiments of the solutions, slurries, and suspensionsdisclosed herein, the crude solid material comprising the compound offormula (I) is less than 70% pure, less than 75% pure, less than 80%pure, less than 85% pure, or less than 90% pure with respect to thecompound of formula (I). In certain embodiments, the crude solidmaterial comprising the compound of formula (I) is less than 90% purewith respect to the compound of formula (I). In certain embodiments, thecrude solid material comprises about 70% to about 90% compound offormula (I). In some embodiments, the purity of the crude solid materialis about 70% to about 90% with respect to the compound of formula (I).

In certain embodiments, after crystallization, the compound of formula(I) is substantially pure. In some embodiments, the crystalline form ofthe compound of formula (I) is greater than 90% pure. In someembodiments, the purity of the crystalline form of the compound offormula (I) is selected from greater than 90%, greater than 91%, greaterthan 92%, greater than 93%, greater than 94%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98%, and greater than 99%. Insome embodiments, the purity of the crystalline form of the compound offormula (I) is greater than 95%. In some embodiments, the purity of thecrystalline form of the compound of formula (I) is greater than 98%. Insome embodiments, the purity of the crystalline form of the compound offormula (I) is selected from about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, and about 99%.

In certain embodiments, the crystalline compound made by the methods ofthe invention is a solvate, e.g., a hydrate.

In certain embodiments, the crystalline compound made by the methods ofthe invention is a monohydrate.

In certain embodiments, the crystalline compound made by the methods ofthe invention is a dihydrate.

In some embodiments, solvent vapor slowly diffuses into a solid sample.In some embodiments, the solvent vapor is water vapor. In someembodiments, for example to achieve Form 2, the solid sample is Form 1,and solvent is water.

In certain embodiments, the crystalline Form 1 of compound of formula(I), can be substantially free of other crystalline forms of compound offormula (I). In certain embodiments, the crystalline Form 1 of compoundof formula (I), can be substantially free of crystalline Form 2 ofcompound of formula (I).

In certain embodiments, the crystalline Form 2 of compound of formula(I), can be substantially free of other crystalline forms of compound offormula (I). In certain embodiments, the crystalline Form 2 of compoundof formula (I), can be substantially free of crystalline Form 1 ofcompound of formula (I).

In certain embodiments, the mixture comprising the compound of formula(I) is a solution, and the step of crystallizing the compound from themixture comprises bringing the solution to supersaturation to cause thecompound of formula (I) to precipitate out of solution.

In certain embodiments, bringing the mixture comprising the compound offormula (I) to supersaturation comprises the slow addition of ananti-solvent, such as heptanes, hexanes, ethanol, or another polar ornon-polar liquid miscible with an aqueous solution, allowing thesolution to cool (with or without seeding the solution), reducing thevolume of the solution, or any combination thereof. In certainembodiments, the anti-solvent is ethanol, isopropanol, methanol,tetrahydrofuran, 1,4-dioxane, acetonitrile, methyl tert-butyl ether,isopropyl acetate and acetone. In certain embodiments, the anti-solventis ethanol, isopropanol, methanol, tetrahydrofuran, 1,4-dioxane,acetonitrile and acetone. In certain embodiments, the anti-solvent isethanol. In certain embodiments, bringing the mixture comprising thecompound of formula (I) to supersaturation comprises adding ananti-solvent, cooling the solution to ambient temperature or lower, andreducing the volume of the solution, e.g., by evaporating solvent fromthe solution. In certain embodiments, allowing the solution to cool maybe passive (e.g., allowing the solution to stand at ambient temperature)or active (e.g., cooling the solution in an ice bath or freezer).

In certain embodiments, the preparation method further comprisesisolating the crystals, e.g., by filtering the crystals, by decantingfluid from the crystals, or by any other suitable separation technique.In further embodiments, the preparation method further comprises washingthe crystals.

In certain embodiments, the preparation method further comprisesinducing crystallization. The method can also comprise drying thecrystals, for example under reduced pressure. In certain embodiments,inducing precipitation or crystallization comprises secondarynucleation, wherein nucleation occurs in the presence of seed crystalsor interactions with the environment (crystallizer walls, stirringimpellers, sonication, etc.).

In other embodiments, the solvent is acetonitrile, diethyl ether,N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide(DMSO), dichloromethane, ethanol, ethyl acetate, heptanes, hexanes,isopropyl acetate, methanol, methylethyl ketone, N-methyl-2-pyrrolidone(NMP), tetrahydrofuran, toluene, 2-propanol (isopropanol), 1-butanol,water, or any combination thereof. In some embodiments, the solvent isdichloromethane. In some preferred embodiments, for example to achieveForm 1, the solvent is tetrahydrofuran.

In some embodiments, the anti-solvent is selected from tetrahydrofuran,methanol, isopropanol, diethyl ether, ethanol, 1,4-dioxane,acetonitrile, and acetone. In some embodiments, to an aqueous solutioncomprising the compound of formula (I) is slowly added an anti-solvent.In certain preferred embodiments, for example to achieve Form 2, anaqueous solution comprising Form 1 is combined with an anti-solventselected from tetrahydrofuran, methanol, isopropanol, diethyl ether,ethanol, 1,4-dioxane, acetonitrile, and acetone.

In some embodiments, the anti-solvent is selected from tetrahydrofuran,methanol, isopropanol, 1,4-dioxane, acetonitrile, and acetone. In someembodiments, an anti-solvent slowly diffuses into an aqueous solutioncomprising the compound of formula (I). In certain preferredembodiments, for example to achieve Form 2, an aqueous solutioncomprising Form 1 is diffused with an anti-solvent selected fromtetrahydrofuran, methanol, isopropanol, 1,4-dioxane, acetonitrile, andacetone.

In some embodiments, a slurry comprising the compound of formula (I) anda solvent were mixed before isolating the solids. In some embodiments,the isolation of the solids is by filtration or centrifugation. In someembodiments, to achieve Form 2, the slurry comprising Form 1 was mixedwith a solvent selected from acetonitrile, anisole, dichloromethane,ethanol, isopropyl acetate, methyl tert-butyl ether (MTBE), n-heptane,tetrahydrofuran, water, and mixtures thereof.

In some embodiments, to achieve Form 2, the slurry comprising Form 1 wasmixed with a solvent selected from acetonitrile, ethanol, methyltert-butyl ether (MTBE), water, and mixtures thereof.

In some embodiments, to achieve Form 2, the slurry comprising compoundwas mixed with a solvent selected from acetonitrile, methyl tert-butylether (MTBE), water, and mixtures thereof.

In certain embodiments, the solvent is a mixture comprising water. Insome preferred embodiments, the solvent is a mixture comprising waterand ethanol, isopropanol, methanol, or tetrahydrofuran. In certainpreferred embodiments, for example to achieve Form 2, the solvent is amixture comprising water and tetrahydrofuran. In other embodiments thesolvent is a mixture comprising water, and other solvents selected fromethanol, isopropanol, methanol, tetrahydrofuran, 1,4-dioxane,acetonitrile, and acetone.

In certain preferred embodiments, the solvent mixture is selected fromwater/tetrahydrofuran (H₂O/THF), water/methanol (H₂O/MeOH),water/isopropanol (H₂O/IPA), water/ethanol (H₂O/EtOH), water/1,4-dioxane(H₂O/1,4-dioxane), water/acetonitrile (H₂O/ACN), or water/Acetone(H₂O/Acetone).

In some embodiments, the solvent is a mixture comprising EtOH:H₂O in avolume-to-volume ratio selected from 19:2, 5:1, 2:1, 1:1, and 1:9. Insome embodiments, the solvent is a mixture comprising ethanol and waterto which additional ethanol or a mixture of ethanol and water is added.In some embodiments, the mixture comprises the compound of formula (I)and a solvent of 2:1 EtOH:H₂O (v/v), then a mixture of 19:2 EtOH:H₂O isadded for crystallization.

In some embodiments, crystallization is aided by seeding or seedloading, that is adding seed crystals to the mixture. In someembodiments, the seed crystals are added at a weight percentage of thetotal mixture selected from about 1 wt %, about 2 wt %, about 3 wt %,about 4 wt %, about 5 wt %, about 6 wt %, about 7 wt %, about 8 wt %,about 9 wt %, and about 10 wt %. In some embodiments, the seed crystalsare added at a weight percentage of the total mixture selected fromabout 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about 5 wt %,and about 6 wt %. In some embodiments, the seed crystals are added at aweight percentage of the total mixture selected from 3 wt %, 4 wt %, and5 wt %.

In some embodiments, the seed crystals are formula (I) Form 2 seedcrystals. In certain embodiments, the seed crystals are milled.

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, ethanol, heptanes,hexanes, methanol, tetrahydrofuran, toluene, water, or a combinationthereof.

As used herein, “anti-solvent” means a solvent in which the compoundcrystals are insoluble, minimally soluble, or partially soluble. Inpractice, the addition of an anti-solvent to a solution in which thesalt crystals are dissolved reduces the solubility of the salt crystalsin solution, thereby stimulating precipitation of the salt. In certainembodiments, the crystals are washed with a combination of anti-solventand the organic solvent. In certain embodiments, the anti-solvent iswater, while in other embodiments it is an alkane solvent, such ashexane or pentane, or an aromatic hydrocarbon solvent, such as benzene,toluene, or xylene. In certain embodiments, the anti-solvent ismethanol.

In certain embodiments, washing the crystals comprises washing thecrystalline compound of formula (I) with a solvent or a mixture of oneor more solvents, which are described above. In certain embodiments, thesolvent or mixture of solvents is cooled prior to washing.

In certain embodiments, the methods of making the crystalline forms ofthe compound of formula (I) are used to remove one or more impuritiesfrom a sample of the compound of formula (I). In certain embodiments,the crystallization methods described herein are used for purifying thecompound of formula (I), e.g., as a final purification step in themanufacture of the compound.

In certain embodiments, the compound of formula (I) is purified bycrystallization. In some embodiments, purification of the compound offormula (I) does not use high-performance liquid chromatography (HPLC),including preparative HPLC. In some embodiments, purification of thecompound of formula (I) by crystallization is scalable. Advantages ofpurification by crystallization include, but are not limited to, removalof soluble impurities, ease of purification process, applicability tolarge scale synthesis, acceptable yields, and high product purity.

In some embodiments, the crystalline formula (I) Form 2 was the formwith greater stability. In some embodiments, the crystalline formula (I)Form 2 is more stable than the crystalline formula (I) Form 1 in water.In some embodiments, the temperature was below about 20° C., below about15° C., below about 10° C., below about 5° C., or below about 0° C. Insome embodiments, the temperature was about 20° C., about 15° C., about10° C., about 5° C., or about 0° C. In some embodiments, the temperaturewas about 10° C., about 9° C., about 8° C., about 7° C., about 6° C.,about 5° C., about 4° C., about 3° C., about 2° C., about 1° C., about0° C., about −1° C., or about −2° C. In some embodiments, thetemperature was about 10° C.

In some embodiments, the conversion to crystalline formula (I) Form 2was complete in about 4 h, in about 8 h, in about 12 h, in about 16 h,in about 20 h, in about 1 day, in about 2 days, in about 3 days, inabout 4 days, in about 5 days, in about 6 days, in about 7 days. In someembodiments, the conversion to crystalline formula (I) Form 2 wascomplete in about 2 h, in about 3 h, in about 4 h, in about 5 h, inabout 6 h, in about 7 h, in about 8 h, in about 9 h, in about 10 h, inabout 11 h, in about 12 h, in about 13 h, in about 14 h, in about 15 h,in about 16 h, in about 17 h, in about 18 h, in about 19 h, in about 20h, in about 21 h, in about 22 h, in about 23 h, in about 24 h, in about25 h, or in about 26 h.

Uses of Crystal Forms of the Compound of Formula (I)

The compound of formula (I) is a 3-substituted 1,2,4-oxadiazole compoundhaving the following structure,

Functional “exhaustion” (immune dysfunction) among T and B cell subsetsis a well-described feature of chronic viral infections, such ashepatitis B and C and HIV viruses. T cell exhaustion was initiallydescribed for CD8 T cells in mice chronically infected with lymphocyticchoriomeningitis virus clone 13. In the lymphocytic choriomeningitisvirus mouse model, repeated antigen stimulation through the T cellantigen receptor drives the sustained expression of T cell inhibitoryreceptors, including programmed cell death-1 (PD-1) and lymphocyteactivationgene-3 (LAG-3), on virus-specific CD8 T cells (J. Illingworthet al., J. Immunol. 2013, 190(3): 1038-1047).

Thus, diseases modulated by an immune response including, but notlimited to, cancer, immune disorders, immunodeficiency disorders,inflammatory disorders, infectious diseases, and transplant rejection,can be treated by administering an immunomodulator, such as the compoundof formula (I), and compositions disclosed herein. 3-substituted1,2,4-oxadiazole compounds act as immunomodulators.

In certain embodiments, the compound of formula (I) modulates an immuneresponse in a cell.

In other embodiments, the present disclosure provides a method ofmodulating an immune response in a cell, comprising contacting the cellwith a composition comprising a crystalline form of the compound offormula (I), according to any of the above embodiments. In someembodiments, the present disclosure provides a method of modulating animmune response in a cell, comprising contacting the cell with acomposition comprising a crystalline form of the compound of formula(I), according to any of the above embodiments.

In certain embodiments, the present disclosure provides uses of acrystalline form of the compound of formula (I) for the preparation of amedicament, e.g., for the treatment of cancer, immune disorders,immunodeficiency disorders, inflammatory disorders, infectious diseases,and transplant rejection.

In accordance with any of the foregoing embodiments, in certainembodiments, contacting the cell occurs in a subject in need thereof,thereby treating a disease or disorder selected from cancer, immunedisorders, immunodeficiency disorders, inflammatory disorders,infectious diseases, and transplant rejection.

In certain embodiments, the present disclosure provides methods fortreating cancer, wherein the method comprises administration of atherapeutically effective amount of a composition comprising acrystalline form of the compound of formula (I) the subject in needthereof.

In certain embodiments, the present disclosure provides methods forinhibiting growth of tumor cells and/or metastasis by administering atherapeutically effective amount of a composition comprising acrystalline form of the compound of formula (I) to the subject in needthereof.

Representative tumor cells include cells of a cancer such as, but notlimited to, blastoma (e.g., glioblastoma), breast cancer (e.g., breastcarcinoma, primary ductal carcinoma, triple negative breast cancer,estrogen receptor positive (ER+), progesterone receptor positive (PR+),and/or human epidermal growth factor receptor 2 positive (HER2+)),epithelial cancer (e.g., carcinomas), colon cancer, lung cancer (e.g.,small cell lung cancer, non-small cell lung cancer (NSCLC), lungadenocarcinoma, and lung squamous cell carcinoma), melanoma (e.g.,cutaneous melanoma, ocular melanoma, cutaneous or intraocular malignantmelanoma, and lymph node-associated melanoma), prostate cancer (e.g.,prostate adenocarcinoma), renal cancer (e.g., renal cell cancer (RCC)and kidney cancer), bone cancer (e.g., osteosarcoma), pancreatic cancer(e.g., pancreatic adenocarcinoma), skin cancer, cancer of the head orneck (e.g., head and neck squamous cell carcinoma), uterine cancer,ovarian cancer (e.g., ovarian carcinoma), colorectal cancer (e.g.,microsatellite instability high colorectal cancer and colorectaladenocarcinoma), rectal cancer, cancer of the anal region, cancer of theperitoneum, stomach cancer (e.g., gastric carcinoma and gastrointestinalcancer), testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, cervical cancer (e.g., carcinoma of the cervix),vaginal cancer (e.g., carcinoma of the vagina), vulval cancer (e.g.,carcinoma of the vulva), cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, thyroid cancer (e.g., cancerof the thyroid gland), cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma (e.g., sarcoma of soft tissue and Kaposi'ssarcoma), cancer of the urethra, cancer of the penis, chronic or acuteleukemia,(e.g., acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, Hairy cellleukemia, and chronic myeloblastic leukemia,), solid tumors ofchildhood, Hodgkin's lymphoma (HL) (e.g., lymphocyte-rich (LRCHL),nodular sclerosis (NSHL), mixed cellularity (MCHL) and lymphocytedepleted (LDHL)), B-cell lymphomas (e.g., diffuse large B-cell lymphoma(DLBCL)), non-Hodgkin's lymphoma (NHL) (e.g., low grade/follicularnon-Hodgkin's lymphoma, small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, mantle celllymphoma), AIDS-related lymphoma, cutaneous T-cell lymphoma (e.g.,mycosis fundoides) and Waldenstrom's Macroglobulinemia, post-transplantlymphoproliferative disorder (PTLD), lymphocytic lymphoma, primary CNSlymphoma, and T-cell lymphoma), mesothelioma, thymic carcinoma, myeloma(e.g., multiple myeloma), cancer of the bladder (e.g., bladdercarcinoma), cancer of the ureter, carcinoma of the renal pelvis, livercancer (e.g., hepatocellular cancer, hepatic carcinoma, hepatoma),pancreatic cancer, post-transplant lymphoproliferative disorder (PTLD),neoplasm of the central nervous system (CNS), tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer,salivary gland carcinoma, squamous cell cancer, abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), Meigs' syndrome, Merkel cell carcinoma,environmentally induced cancers (including those induced by asbestos),and combinations of said cancers.

In other embodiments, for example, the tumor cells may include cells ofa cancer selected from prostate cancer, melanoma, breast cancer, coloncancer, prostate cancer, lung cancer, renal cancer, pancreatic cancer,gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma,thyroid cancer, thymic carcinoma, sarcoma, glioblastoma, chronic oracute leukemia, lymphoma, myeloma, Merkel cell carcinoma, epithelialcancer, colorectal cancer, vaginal cancer, cervical cancer, ovariancancer, and cancer of the head and neck.

In other embodiments, for example, the tumor cells may include cells ofa cancer selected from melanoma, triple negative breast cancer,non-small cell lung cancer, renal cell carcinoma, pancreatic cancer,gastric carcinoma, bladder cancer, mesothelioma, Hodgkins's lymphoma,cervical cancer, ovarian cancer, and head and neck squamous cellcarcinoma.

In some embodiments, the tumor cells are cells of a cancer selected fromsmall cell lung cancer, multiple myeloma, bladder carcinoma, primaryductal carcinoma, ovarian carcinoma, Hodgkin's lymphoma, gastriccarcinoma, acute myeloid leukemia, and pancreatic cancer.

In other embodiments, the tumor cells are cells of a cancer selectedfrom carcinoma of the endometrium, ovarian cancer, Hodgkin's lymphoma,non-Hodgkin's lymphoma, and chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, andmultiple myeloma.

In some embodiments, the tumor cells are cells of a cancer selected fromprostate adenocarcinoma, lung adenocarcinoma, lung squamous cellcarcinoma, pancreatic adenocarcinoma, breast cancer and colorectaladenocarcinoma. In certain embodiments, tumor cells are from breastcancer. In some embodiments, the tumor cells are from a breast cancerselected from triple negative breast cancer, estrogen receptor positive(ER+), progesterone receptor positive (PR+), and/or human epidermalgrowth factor receptor 2 (HER2+). In other embodiments, the tumor cellsare from a PAM50+ breast cancer assay panel (Parker, J. S., et al., J.Clin. Oncol., 2009, 27(8): 1160-1167), breast cancer selected fromluminal A, luminal B, HER2-enriched, basal-like and normal-like.

In some embodiments, the tumor cells are cells of a cancer selected fromtriple negative breast cancer, microsatellite instability highcolorectal cancer, gastric carcinoma, mesothelioma, pancreatic cancer,and cervical cancer.

In some embodiments, the tumor cells are, and/or the subject is, naïveto immunooncology therapy. Immunooncology uses the subject's immunesystem to help fight cancer. For example, an immunooncology therapyincludes, but is not limited to, atezolizumab (human monoclonal antibodythat targets PD-L1), avelumab (human monoclonal antibody that targetsPD-L1), brentuximab vedotin (antibody-drug conjugate that targets CD30),durvalamab (human monoclonal antibody that targets PD-L1), ipilimumab(human monoclonal antibody that targets CTLA-4), nivolumab (humanmonoclonal antibody that targets PD-L1), pembrolizumab (also referred toas lambrolizumab, human monoclonal antibody that targets PD-L1),tremelimumab (human monoclonal antibody that targets CTLA-4), CT-011(antibody that targets PD-1), MDX-1106 (antibody that targets PD-1),MK-3475 (antibody that targets PD-1), YW243.55.S70 (antibody thattargets PD-L1), MPDL3280A(antibody that targets PD-L1), MDX-1105(antibody that targets PD-L1), and MEDI4736 (antibody that targetsPD-L1). In some embodiments, the immunooncology therapy is selected froman anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody,an anti-PD-L2 antibody, an anti-TIGIT antibody (e.g., antibodiesdisclosed in WO 2015/009856).

In other embodiments, the tumor cells are, and/or the subject isresponsive to immune checkpoint therapy. In some embodiments, the cancerhas shown response to anti-PD1 therapy. For example, the cancer mayinclude non-small cell lung cancer (NSCLC), melanoma, renal cell cancer(RCC), cancer of the bladder, Hodgkin's lymphoma, and head and necksquamous cell carcinoma.

Other embodiments of the present disclosure provide a method oftreatment of infection.

Still other embodiments of the present disclosure provide a method oftreatment of infection comprising administration of a therapeuticallyeffective amount of a composition comprising a crystalline form of thecompound of formula (I) to the subject in need thereof.

In certain embodiments, the present disclosure provides uses of acrystalline form of the compound of formula (I) for the preparation of amedicament for the treatment of infectious disease, as well as methodsof administering a therapeutically effective amount of a compositioncomprising a crystalline form of the compound of formula (I) for thetreatment of infectious disease.

In some embodiments, the infectious disease is bacterial infection,viral infection, fungal infection, or parasitic infection, as well asmethods of administering a therapeutically effective amount of acomposition comprising a crystalline form of the compound of formula (I)for the treatment of bacterial infection, viral infection, fungalinfection, or parasitic infection.

In some embodiments, for example, bacterial infection may be caused byat least one bacterium selected from anthrax, Bacilli, Bordetella,Borrelia, botulism, Brucella, Burkholderia, Campylobacter, Chlamydia,cholera, Clostridium, Conococcus, Corynebacterium, diptheria,Enterobacter, Enterococcus, Erwinia, Escherichia, Francisella,Haemophilus, Heliobacter, Klebsiella, Legionella, Leptospira,leptospirosis, Listeria, Lyme's disease, meningococcus, Mycobacterium,Mycoplasma, Neisseria, Pasteurella, Pelobacter, plague, Pneumonococcus,Proteus, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella,Staphylococcus, Streptococcus, tetanus, Treponema, Vibrio, Yersinia andXanthomonas.

In other embodiments, for example, viral infection may be caused by atleast one virus selected from Adenoviridae, Papillomaviridae,Polyomaviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Parvoviridae,Astroviridae, Caliciviridae, Picornaviridae, Coronoviridae,Flaviviridae, Retroviridae, Togaviridae, Arenaviridae, Bunyaviridae,Filoviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, andReoviridae. In certain embodiments, the virus may be arboviralencephalitis virus, adenovirus, herpes simplex type I, herpes simplextype 2, Varicella-zoster virus, Epstein-barr virus, cytomegalovirus,herpesvirus type 8, papillomavirus, BK virus, coronavirus, echovirus, JCvirus, smallpox, Hepatitis B, bocavirus, parvovirus B19, astrovirus,Norwalk virus, coxsackievirus, Hepatitis A, poliovirus, rhinovirus,severe acute respiratory syndrome virus, Hepatitis C, yellow fever,dengue virus, West Nile virus, rubella, Hepatitis E, humanimmunodeficiency virus (HIV), human T-cell lymphotropic virus (HTLV),influenza, guanarito virus, Junin virus, Lassa virus, Machupo virus,Sabia virus, Crimean-Congo hemorrhagic fever virus, ebola virus, Marburgvirus, measles virus, molluscum virus, mumps virus, parainfluenza,respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipahvirus, rabies, Hepatitis D, rotavirus, orbivirus, coltivirus, vacciniavirus, and Banna virus.

In other embodiments, for example, fungal infection may be selected fromthrush, Aspergillus (fumigatus, niger, etc.), Blastomyces dermatitidis,Candida (albicans, krusei, glabrata, tropicalis, etc.), Coccidioidesimmitis, Cryptococcus (neoformans, etc.), Histoplasma capsulatum,Mucorales (mucor, absidia, rhizophus), Paracoccidioides brasiliensis,sporotrichosis, Sporothrix schenkii, zygomycosis, chromoblastomycosis,lobomycosis, mycetoma, onychomycosis, piedra pityriasis versicolor,tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea favosa,tinea nigra, tinea pedis, otomycosis, phaeohyphomycosis, andrhinosporidiosis.

In some embodiments, for example, parasitic infection may be caused byat least one parasite selected from Acanthamoeba, Babesia microti,Balantidium coli, Entamoeba hystolytica, Giardia lamblia,Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatidarhodesiense, Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana,Leishmania braziliensis, Leishmania tropica, Leishmania donovani,Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Plasmodium falciparum, Pneumocystis carinii, Trichomonasvaginalis, Histomonas meleagridis, Secementea, Trichuris trichiura,Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale,Naegleria fowleri, Necator americanus, Nippostrongylus brasiliensis,Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis,blood flukes, liver flukes, intestinal flukes, lung flukes, Schistosomamansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciolahepatica, Fasciola gigantica, Heterophyes heterophyes, and Paragonimuswestermani.

The term “subject” includes mammals (especially humans) and otheranimals, such as domestic animals (e.g., household pets including catsand dogs) and non-domestic animals (such as wildlife).

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence or frequency of the disorder or condition in the treatedsample relative to an untreated control sample, or delays the onset orreduces the severity of one or more symptoms of the disorder orcondition relative to the untreated control sample. Thus, prevention ofcancer includes, for example, reducing the number of detectablecancerous growths in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/or delayingthe appearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

Pharmaceutical Compositions

In certain embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a crystalline form of the compound of formula (I)as disclosed herein, optionally admixed with a pharmaceuticallyacceptable carrier or diluent.

The present disclosure also provides methods for formulating thedisclosed crystalline forms of the compound of formula (I) forpharmaceutical administration.

The compositions and methods of the present disclosure may be utilizedto treat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a crystalline form of the compound of formula(I) of the disclosure and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil, or injectable organic esters. In a preferredembodiment, when such pharmaceutical compositions are for humanadministration, particularly for invasive routes of administration(i.e., routes, such as injection or implantation, that circumventtransport or diffusion through an epithelial barrier), the aqueoussolution is pyrogen-free, or substantially pyrogen-free. The excipientscan be chosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophile for reconstitution, powder, solution, syrup, suppository,injection or the like. The composition can also be present in atransdermal delivery system, e.g., a skin patch. The composition canalso be present in a solution suitable for topical administration, suchas an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acrystalline form of the compound of formula (I) of the disclosure. Suchphysiologically acceptable agents include, for example, carbohydrates,such as glucose, sucrose or dextrans, antioxidants, such as ascorbicacid or glutathione, chelating agents, low molecular weight proteins orother stabilizers or excipients. The choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable agent,depends, for example, on the route of administration of the composition.The preparation of pharmaceutical composition can be a self-emulsifyingdrug delivery system or a self-microemulsifying drug delivery system.The pharmaceutical composition (preparation) also can be a liposome orother polymer matrix, which can have incorporated therein, for example,a crystalline form of the compound of formula (I) of the disclosure.Liposomes, for example, which comprise phospholipids or other lipids,are nontoxic, physiologically acceptable and metabolizable carriers thatare relatively simple to make and administer.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a crystallineform of the compound of formula (I) of the disclosure, with the carrierand, optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a compound of the present disclosure with liquid carriers,or finely divided solid carriers, or both, and then, if necessary,shaping the product.

Formulations of the disclosure suitable for oral administration may bein the form of capsules (including sprinkle capsules and gelatincapsules), cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), lyophile, powders, granules,or as a solution or a suspension in an aqueous or non-aqueous liquid, oras an oil-in-water or water-in-oil liquid emulsion, or as an elixir orsyrup, or as pastilles (using an inert base, such as gelatin andglycerin, or sucrose and acacia) and/or as mouth washes and the like,each containing a predetermined amount of a compound of the presentdisclosure as an active ingredient. Compositions or compounds may alsobe administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present disclosure to the body. Suchdosage forms can be made by dissolving or dispersing the active compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this disclosure.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference in its entirety. If desired, liquid ophthalmic formulationshave properties similar to that of lacrimal fluids, aqueous humor orvitreous humor or are compatible with such fluids. A preferred route ofadministration is local administration (e.g., topical administration,such as eye drops, or administration via an implant).

A suppository also is contemplated as being within the scope of thisdisclosure.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this disclosure, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the crystalline form of the compound of formula (I) ofthe disclosure. A larger total dose can be delivered by multipleadministrations of the agent. Methods to determine efficacy and dosageare known to those skilled in the art (Isselbacher et al. (1996)Harrison's Principles of Internal Medicine 13 ed., 1814-1882, hereinincorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the disclosure will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentdisclosure, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES

Analytical Methods

X-Ray Powder Diffraction

X-Ray Powder Diffraction patterns were collected on an Empyreandiffractometer or an X'Pert3 diffractometer using Cu Kα radiation (45kV, 40 mA).

The details of the data collection are summarized in Table 3:

TABLE 3 X-Ray Powder Diffraction Parameters Instrument PANalytical ModelEmpyrean X′ Pert3 Empyrean (Reflection (Reflection (Transmission Mode)Mode) Mode) X-Ray wavelength Cu, kα, Kα1 (Å): 1.540598, Kα2 (Å):1.544426 Kα2/Kα1 intensity ratio: 0.50 X-Ray tube setting 45 kV, 40 mADivergence slit Automatic Fixed ⅛° Fixed ½° Scan mode Continuous Scanrange (°2TH) 3°-40° Scan step time [s] 17.8 46.7 33.02 Step size (°2TH)0.0167 0.0263 0.0167 Test Time (s) 5 min 30 s 5 min 04 s 10 min 11sHPLC

Purity analysis was performed on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.04.03 using the method detailed below in Table 4.

TABLE 4 HPLC Parameters Parameter Value Type of method Reversed phasewith gradient elution Sample Preparation Diluent, acetonitrile/H₂O = 1:1Column ZIC-HILIC, 250 × 4.6 mm, 5 μm Column Temperature 30° C. InjectionVolume 10 μL Detector Wavelength, Bandwidth UV at 210 nm Flow Rate 1.0mL/min Mobile Phase A 10 mM KH₂PO₄ in H₂O Mobile Phase B acetonitrileGradient Timetable Time (min) % Mobile Phase A 0.0 80 2.0 80 20.0 6020.1 80 30.0 80Thermogravimetric Analysis and Differential Scanning Calorimetry:

Thermogravimetric analysis (TGA) data were collected using a TAQ500/Q5000 TGA from TA Instruments. Differential scanning calorimetry(DSC) was performed using a TA Q200/Q2000 DSC from TA Instruments.Method parameters are provided in Table 5 below.

TABLE 5 TGA and DSC Parameters Parameters TGA DSC Method Ramp Ramp PanPlatinum, open Aluminum plate, crimped Temperature RT-Target Temperature25° C.-Target Temperature Ramp rate 10° C./min 10° C./min Purge gas N₂N₂PXRD Determination of Formula (I) as Reported in WO 2015/033299

The PXRD determination of the compound of Formula (I) was obtained usingthe procedure described in Example 4 of WO 2015/033299, which depictsthe compound to be amorphous. The PXRD is shown in FIG. 8. The contentsof WO 2015/033299 are hereby incorporated by reference in theirentirety.

Example 1 Synthesis of Form 1

A. Crystallization from Tetrahydrofuran

Formula (I) Form 1 exists at ambient conditions upon short term storage(i.e., less than 8 d). A mixture of formula (I) Forms 1 and 2 (16 mg)was weighed into a glass vial, and tetrahydrofuran (THF, 0.5 mL) wasadded. The resulting slurry was stirred at room temperature for about 4d. The wet solids were analyzed by XRPD in transmission mode andconfirmed to be crystalline formula (I) Form 1, as shown by XRPDanalysis (FIG. 1).

B. Crystallization from an Ethanol/Water Mixture

About 68.0 g of amorphous compound of formula (I) was dissolved in aminimum amount of water, and ethanol was added to the solution to obtaina white precipitate. The solid was isolated by filtration. The solid waswashed with ethanol followed by a wash with diethyl ether. The solid wasdried under high vacuum and confirmed to be formula (I) Form 1 by XRPDanalysis.

Example 2 Synthesis of Form 2

A. Solid Vapor Diffusion

Formula (I) Form 1 (approximately 15.3 mg) was weighed into a 3-mL vial,which was then placed into a 20-mL vial with 4.0 mL of water. The 20-mLvial was sealed with a cap and kept at RT for a week allowing solventvapor to interact with the sample. The obtained solid was shown by XRPDanalysis to be crystalline formula (I) Form 2.

B. Anti-Solvent Addition

About 15.0 mg of formula (I) Form 1 sample was dissolved in 0.3 mL ofH₂O in a 20-mL glass vial and stirred at RT. The correspondinganti-solvent (e.g., THF, MeOH, IPA, EtOH, 1,4-dioxane, ACN or Acetone)was added to the above aqueous solution till precipitate appeared or thetotal volume reached 15.0 mL. The solids were isolated by centrifugationand confirmed to be formula (I) Form 2 by XRPD analysis.

TABLE 6 Exemplary Solvent Systems to Synthesize Formula (I) Form 2 byAnti-Solvent Addition No. Methods Solvent System 1 Anti-solvent additionH₂O/THF 2 H₂O/MeOH 3 H₂O/IPA 4 H₂O/EtOH 5 H₂O/1,4-dioxane 6 H₂O/ACN 7H₂O/AcetoneC. Solution Vapor Diffusion

About 15.0 mg of formula (I) Form 1 was dissolved in 0.3 mL of H₂O in a3-mL glass vial at RT. The clear solutions in uncapped, open vials werethen placed into a 20-mL vial with 4.0 mL of the anti-solvent (e.g.,THF, MeOH, IPA, EtOH, 1,4-dioxane, ACN or Acetone). The 20-mL vial wassealed with a cap and kept at RT allowing interaction between vapor andthe solution. The solids were confirmed to be formula (I) Form 2 by XRPDanalysis.

TABLE 7 Exemplary Solvent Systems to Synthesize Formula (I) Form 2 byVapor Diffusion No. Methods Solvent System 1 Solution vapor diffusionH₂O/thf 2 H₂O/MeOH 3 H₂O/IPA 4 H₂O/ACN 5 H₂O/1,4-dioxane 6 H₂O/acetoneD. Slurry

About 20.0 mg of formula (I) Form 1 was weighed into a glass vial, and0.5 mL of corresponding solvent (as shown in Table 8) was added. Afterslurry at the assigned temperature (mentioned below in Table 6) forabout 3 days, the solids were isolated by centrifugation and confirmedto be formula (I) Form 2 by XRPD analysis (FIG. 2)

TABLE 8 Exemplary Solvent Systems to Synthesize Formula (I) Form 2 bySlurry No. Methods Solvent System, v:v 1 Slurry at RT EtOH 2 Anisole 3MTBE 4 n-heptane 5 DCM 6 EtOH/H₂O, 97:3, a_(w) = 0.2 7 EtOH/H₂O,92.7:7.3, a_(w) = 0.4 8 EtOH/H₂O, 86:14, a_(w) = 0.6 9 EtOH/H₂O, 71:29,a_(w) = 0.8 10 ACN/MTBE, 1:1 11 Slurry at 50° C. EtOH 12 IPAc 13THF/IPAc, 1:1 v:v = vol./vol; aw = water activity at 25° C.E. Anti-Solvent Addition Using Water/Acetonitrile Mixture

The compound was dissolved in one volume of water and added 10 vol. ofacetonitrile to that and stirred for 12 hrs. The white solid wasprecipitated out. The precipate was filtered and washed with 1% H₂O inAcetonitrile (2 vol) and washed with Acetonitrile (2 vol). Thepreceipitate was finally washed with MTBE (10 vol) and dried under highvacuum for 10-15 h. The solids were confirmed to be formula (I) Form 2by XRPD analysis (FIG. 2).

F. Liquid Vapor Diffusion

About 15.0 mg of formula (I) Form 1 was weighed into a 3 mL glass vialwith the addition of 0.3 mL H₂O (solvent). After vortexing, thesuspension was filtered by Polytetrafluoroethylene (PTFE) filteringmembrane, and the solution was transferred to a new 3 mL glass vial.Then the uncapped, open vial was placed in a 20 mL glass vial with 4 mLof 1,4-dioxane (anti-solvent), and the system was kept at roomtemperature for liquid vapor diffusion. After 70 days at roomtemperature, rod-like crystals suitable for single crystal X-raydiffraction were obtained.

Example 3 Thermogravimetric Analysis and Differential ScanningCalorimetry of Formula (I)

Thermogravimetric analysis (TGA) of formula (I) Form 1 showed a weightloss of 5.2% up to 80° C., which is consistent with a monohydrate form.Differential scanning calorimetry (DSC) showed two overlapped endothermsat 125.6° C. and 133.1° C. (peak temperature) beforemelting/decomposition with an onset at 184.1° C. (FIG. 3).

TGA of formula (I) Form 2 showed a two-step weight loss of 9.6% up to80° C., which is consistent with a dihydrate form. DSC showed twoendotherms at 47.2° C. and 72.9° C. (peak temperature) beforemelting/decomposition with an onset at 184.3° C. (FIG. 4).

Example 4 Stability and Forced Degradation Study for Crystalline Formula(I)

Solid Stability

Samples of formula (I) crystalline Form 2 were stored as solids at 25°C./60% Relative Humidity (RH), 30° C./˜56%, and 40° C./75% RH for 7-8days. The samples were prepared in duplicate with an offset of 2 weeks.Each replicate was stored in a different container.

Formula (I) crystalline Form 2 remained unchanged in terms of solid formand particle morphology after storage for 7-8 days at 25° C./60% RH, 30°C./˜56% RH, and 40° C./75% RH. See FIG. 5.

Example 5 Solubility Measurement for Crystalline Formula (I)

Equilibrium solubility of formula (I) Form 2 was measured in the waterat 20° C. and 30° C. All samples were equilibrated at temperature for 6hrs, and the solubility of supernatant was measured by HPLC, while thesolids were checked by XRPD. (See Table 9.)

TABLE 9 Equilibrium Solubility of Crystal Form 2 of Formula (I) Temp.Solubility Starting Form Solvent (° C.) Final Form (mg/mL) Form 2 Water20 Form 2 147.7 Form 2 30 Form 2 165.5

No form change was observed during the solubility testing for Form 2(FIG. 6).

Example 6 Single Crystal Structure Determination

A single crystal for X-ray diffraction was obtained via a liquid vapordiffusion method from H₂O/1,4-dioxane as described in Example 2F. X-raydiffraction data was collected using a PANalytical instrument. Thestructural information and refinement parameters are given in Table 10.

TABLE 10 Structural Information and Refinement Parameters for CrystalForm 2 of Formula (I) Empirical formula C₁₂H₂₄N₆O₉ Temperature 396.37 KWavelength Cu/Kα (λ = 1.54178 Å) Crystal system, space groupOrthorhombic, P2₁2₁2₁ Unit cell dimensions a = 4.8968(2) Å α = 90° b =15.8532(8) Å β = 90° c = 22.3708(11) Å γ = 90° Volume 1736.64(14) Å³ Z,Calculated density 4, 1.516 g/cm³ 2 Theta range for data collection6.834 to 136.702° Reflections collected/Independent 20428/3176 [R(int) =0.1237] reflections Completeness 99.97% Data/restraints/parameters3176/0/321 Goodness-of-fit on F² 1.034 Final R indices [I > 2sigma(I)]R₁ = 0.0429, wR₂ = 0.1052 Largest diff. peak and hole 0.29/−0.33 e · Å⁻³Flack parameter 0.10(17)

Single crystal structural analysis confirmed that the crystallineformula (I) Form 2 is a dihydrate with the asymmetric unit comprised ofone formula (I) molecule and two water molecules (FIG. 7A). One watermolecule forms two hydrogen bonds intramolecularly with two oxygen atoms(O5 and O7) on one formula (I) molecule and intermolecularly forms twohydrogen bonds with each N1 atom on two different formula (I) molecules.This water molecule bridges the negatively and positively charged groupsof the zwitterion. Two additional water molecules form an intermolecularbridge between two N2 atoms. The bond lengths of the C—O/C═O from thecarboxyl group were similar. There were three residual electron densitypeaks assigned as the hydrogen atoms around the N1 atom. Therefore, itis suggested that the formula (I) molecule was a zwitterion in the Form2 crystal (FIG. 7B).

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

The invention claimed is:
 1. A crystalline compound having the structureof formula (I),


2. The crystalline compound of claim 1, wherein the compound issolvated.
 3. The crystalline compound of claim 1, wherein the compoundis a hydrate.
 4. The crystalline compound of claim 3, wherein thecompound is a monohydrate.
 5. The crystalline compound of claim 4,having 2θ values 8.4±0.2, 13.6±0.2, 16.5±0.2, 16.8±0.2, 21.4±0.2, and28.4±0.2.
 6. The crystalline compound of claim 5, having 2θ values8.4±0.2, 13.6±0.2, 16.5±0.2, 16.8±0.2, 19.3±0.2, 20.4±0.2, 21.4±0.2, and28.4±0.2.
 7. The crystalline compound of claim 6, having 2θ values8.4±0.2, 13.6±0.2, 16.5±0.2, 16.8±0.2, 19.3±0.2, 19.9±0.2, 20.4±0.2,21.4±0.2, 24.5±0.2, 26.5±0.2, and 28.4±0.2.
 8. The crystalline compoundof claim 7, having 2θ values 8.4±0.2, 11.5±0.2, 13.6±0.2, 16.5±0.2,16.8±0.2, 19.3±0.2, 19.9±0.2, 20.4±0.2, 21.4±0.2, 21.8±0.2, 24.5±0.2,26.5±0.2, 27.5±0.2, 28.0±0.2, 28.4±0.2, 30.0±0.2, and 32.4±0.2.
 9. Thecrystalline compound of claim 8, having an XRD pattern substantially asshown in FIG.
 1. 10. The crystalline compound of claim 3, wherein thecompound is a dihydrate.
 11. The crystalline compound of claim 10,having 2θ values 12.9±0.2, 13.5±0.2, 15.7±0.2, 17.0±0.2, 29.7±0.2, and33.7±0.2.
 12. The crystalline compound of claim 11, having 2θ values12.9±0.2, 13.5±0.2, 15.7±0.2, 17.0±0.2, 20.3±0.2, 28.9±0.2, 29.7±0.2,and 33.7±0.2.
 13. The crystalline compound of claim 12, having 2θ values12.9±0.2, 13.5±0.2, 15.7±0.2, 17.0±0.2, 19.6±0.2, 20.3±0.2, 26.2±0.2,28.9±0.2, 29.7±0.2, and 33.7±0.2.
 14. The crystalline compound of claim13, having 2θ values 12.9±0.2, 13.5±0.2, 15.7±0.2, 17.0±0.2, 19.1±0.2,19.6±0.2, 20.3±0.2, 21.1±0.2, 21.4±0.2, 26.2±0.2, 27.2±0.2, 28.9±0.2,29.7±0.2, 32.2±0.2, and 33.7±0.2.
 15. The crystalline compound of claim14, having an XRD pattern substantially as shown in FIG.
 2. 16. Apharmaceutical composition comprising the crystalline compound of claim1 and one or more pharmaceutically acceptable excipients.
 17. A methodfor preparing the crystalline compound of claim 1 comprising the stepsof: a) providing a mixture comprising a compound of formula (I) and asolvent; and b) crystallizing the compound of claim 1 from the mixturecomprising the compound of formula (I).
 18. The method of claim 17,wherein the crystalline compound is a solvate.
 19. The method of claim18, wherein the solvate is a hydrate.
 20. The method of claim 17,wherein the compound is monohydrate or a dihydrate.
 21. The method ofclaim 17, wherein the solvent is selected from acetonitrile, anisole,dichloromethane, ethanol, isopropyl acetate, methyl tert-butyl ether(MTBE), n-heptane, tetrahydrofuran, water, and mixtures thereof.
 22. Themethod of claim 17, wherein the mixture further comprises seed crystals.23. The method of claim 22, wherein the seed crystals are added at aweight percentage of the total mixture selected from about 1 wt %, about2 wt %, about 3 wt %, about 4 wt %, about 5 wt %, about 6 wt %, about 7wt %, about 8 wt %, about 9 wt %, and about 10 wt %.
 24. The method ofclaim 22, wherein the seed crystals are formula (I) Form 2 seedcrystals.
 25. The method of claim 17, wherein the purity of thecrystalline form of the compound of formula (I) is selected from about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, and about 99%.